Molecular Characterization of Two Homozygous Factor VII Variants Associated with Intracranial Bleeding

Abstract

Clinical parameters have been extensively studied in factor (F) VII deficiency, but the knowledge of molecular mechanisms of this disease is scarce. We report on three probands with intracranial bleeds at an early age, one of which had concomitant high titer of FVII inhibitor. The aim of the present study was to identify the causative mutations and to elucidate the underlying molecular mechanisms. All nine F7 exons were sequenced in the probands and the closest family members. A homozygous deletion in exon 1, leading to a frame shift and generation of a premature stop codon (p.C10Pfs*16), was found in proband 1. Probands 2 and 3 (siblings) were homozygous for a missense mutation in exon 8, resulting in a glycine (G) to arginine (R) substitution at amino acid 240 (p.G240R). All probands had severely reduced FVII activity (FVII:C < 1 IU/dL). Treatment consisted of recombinant FVIIa and/or plasma concentrate, and proband 1 developed a FVII inhibitor shortly after initiation of treatment. The FVII variants were overexpressed in mammalian cell lines. No FVII protein was produced in cells expressing the p.C10Pfs*16 variant, and the inhibitor development in proband 1 was likely linked to the complete absence of circulating FVII. Structural analysis suggested that the G to R substitution in FVII found in probands 2 and 3 would destabilize the protein structure, and cell studies demonstrated a defective intracellular transport and increased endoplasmic reticulum stress. The molecular mechanism underlying the p.G240R variant could be reduced secretion caused by protein destabilization and misfolding.

Keywords

FVII deficiency - FVII inhibitor - intracranial bleeds - intracellular trafficking - endoplasmic reticulum stress

Author Contributions

E.A. and M.E.C. designed and performed the research, analyzed the data, and wrote the manuscript. M.S., B.S., E.A., P.H.B., B.T., H.G., C.E.H., and N.I. designed and performed the research and analyzed the data. N.I. conceived the study. All authors critically read the manuscript and approved the final version.

Note

Written consent to publish the clinical data was obtained from the parents, as the probands and siblings were minors, and ethical approval was obtained from the data protection officer at the hospital.

^* E.A. and M.E.C. are co-first authors.

Publikationsverlauf

Eingereicht: 19. Februar 2021

Angenommen: 12. März 2021

Accepted Manuscript online:
19. März 2021

Artikel online veröffentlicht:
05. Mai 2021

• References

• 1 Hagen FS, Gray CL, O'Hara P. et al. Characterization of a cDNA coding for human factor VII. Proc Natl Acad Sci U S A 1986; 83 (08) 2412-2416
• 2 McVey JH, Boswell E, Mumford AD, Kemball-Cook G, Tuddenham EG. Factor VII deficiency and the FVII mutation database. Hum Mutat 2001; 17 (01) 3-17
• 3 Thim L, Bjoern S, Christensen M. et al. Amino acid sequence and posttranslational modifications of human factor VIIa from plasma and transfected baby hamster kidney cells. Biochemistry 1988; 27 (20) 7785-7793
• 4 Bolt G, Kristensen C, Steenstrup TD. More than one intracellular processing bottleneck delays the secretion of coagulation factor VII. Thromb Haemost 2008; 100 (02) 204-210
• 5 Bolt G, Steenstrup TD, Kristensen C. All post-translational modifications except propeptide cleavage are required for optimal secretion of coagulation factor VII. Thromb Haemost 2007; 98 (05) 988-997
• 6 Rao LV, Rapaport SI. Activation of factor VII bound to tissue factor: a key early step in the tissue factor pathway of blood coagulation. Proc Natl Acad Sci U S A 1988; 85 (18) 6687-6691
• 7 Mackman N. The role of tissue factor and factor VIIa in hemostasis. Anesth Analg 2009; 108 (05) 1447-1452
• 8 Rosen ED, Chan JCY, Idusogie E. et al. Mice lacking factor VII develop normally but suffer fatal perinatal bleeding. Nature 1997; 390 (6657): 290-294
• 9 Mariani G, Herrmann FH, Bernardi F, Schved JF, Auerswald G, Ingerslev J. Clinical manifestations, management, and molecular genetics in congenital factor VII deficiency: the International Registry on Congenital Factor VII Deficiency (IRF7). Blood 2000; 96 (01) 374
• 10 Mannucci PM, Duga S, Peyvandi F. Recessively inherited coagulation disorders. Blood 2004; 104 (05) 1243-1252
• 11 Mariani G, Bernardi F. Factor VII deficiency. Semin Thromb Hemost 2009; 35 (04) 400-406
• 12 Perry DJ. Factor VII deficiency. Br J Haematol 2002; 118 (03) 689-700
• 13 Borhany M, Pahore Z, Ul Qadr Z. et al. Bleeding disorders in the tribe: result of consanguineous in breeding. Orphanet J Rare Dis 2010; 5: 23
• 14 Girolami A, Bertozzi I, de Marinis GB, Bonamigo E, Fabris F. Activated FVII levels in factor VII Padua (Arg304Gln) coagulation disorder and in true factor VII deficiency: a study in homozygotes and heterozygotes. Hematology 2011; 16 (05) 308-312
• 15 Mariani G, Herrmann FH, Dolce A. et al; International Factor VII Deficiency Study Group. Clinical phenotypes and factor VII genotype in congenital factor VII deficiency. Thromb Haemost 2005; 93 (03) 481-487
• 16 Andersen E, Chollet ME, Myklebust CF. et al. Activation of endoplasmic reticulum stress and unfolded protein response in congenital factor VII deficiency. Thromb Haemost 2018; 118 (04) 664-675
• 17 Tjeldhorn L, Iversen N, Sandvig K, Bergan J, Sandset PM, Skretting G. Protein C mutation (A267T) results in ER retention and unfolded protein response activation. PLoS One 2011; 6 (08) e24009
• 18 Malhotra JD, Miao H, Zhang K. et al. Antioxidants reduce endoplasmic reticulum stress and improve protein secretion. Proc Natl Acad Sci U S A 2008; 105 (47) 18525-18530
• 19 Banner DW, D'Arcy A, Chène C. et al. The crystal structure of the complex of blood coagulation factor VIIa with soluble tissue factor. Nature 1996; 380 (6569): 41-46
• 20 Lillicrap D. The molecular basis of haemophilia B. Haemophilia 1998; 4 (04) 350-357
• 21 Peyvandi F, Mannucci PM, Jenkins PV, Lee A, Coppola R, Perry DJ. Homozygous 2bp deletion in the human factor VII gene: a non-lethal mutation that is associated with a complete absence of circulating factor VII. Thromb Haemost 2000; 84 (04) 635-637
• 22 Shen MC, Lin JS, Lin SW, Yang WS, Lin B. Novel mutations in the Factor VII gene of Taiwanese factor VII-deficient patients. Br J Haematol 2001; 112 (03) 566-571
• 23 Hentze MW, Kulozik AE. A perfect message: RNA surveillance and nonsense-mediated decay. Cell 1999; 96 (03) 307-310
• 24 Shahbazi S. Nonsense-mediated mRNA decay among coagulation factor genes. Iran J Basic Med Sci 2016; 19 (04) 344-349
• 25 Tokgoz H, Caliskan U, Lavigne-Lissalde G, Giansily-Blaizot M. Successful prophylactic use of recombinant activated factor VII (rFVIIa) in a patient with congenital FVII deficiency and inhibitors to FVII. Haemophilia 2012; 18 (01) e25-e27
• 26 McVey JH, Boswell EJ, Takamiya O. et al. Exclusion of the first EGF domain of factor VII by a splice site mutation causes lethal factor VII deficiency. Blood 1998; 92 (03) 920-926
• 27 Giansily-Blaizot M, Lopez E, Viart V. et al. Lethal factor VII deficiency due to novel mutations in the F7 promoter: functional analysis reveals disruption of HNF4 binding site. Thromb Haemost 2012; 108 (02) 277-283
• 28 Giansily-Blaizot M, Aguilar-Martinez P, Briquel ME. et al. Two novel cases of cerebral haemorrhages at the neonatal period associated with inherited factor VII deficiency, one of them revealing a new nonsense mutation (Ser52Stop). Blood Coagul Fibrinolysis 2003; 14 (02) 217-220
• 29 Kruse-Jarres R. Inhibitors: our greatest challenge. Can we minimize the incidence?. Haemophilia 2013; 19 (Suppl. 01) 2-7
• 30 Kempton CL, Soucie JM, Abshire TC. Incidence of inhibitors in a cohort of 838 males with hemophilia A previously treated with factor VIII concentrates. J Thromb Haemost 2006; 4 (12) 2576-2581
• 31 Ramezanpour N, Zaker F, Biswas A, Dorgalaleh A. Inhibitor in congenital factor VII deficiency; a rare but serious therapeutic challenge-a systematic literature review. J Clin Med 2021; 10 (02) 10
• 32 Eshghi P, Tara SZ, Baghaipour MR. et al. Inhibitors against rFVIIa in patients with severe congenital FVII deficiency: a case series. Haemophilia 2019; 25 (05) e345-e349
• 33 Ingerslev J, Christiansen K, Sørensen B. International Registry on Factor VII Deficiency (IRF7) Steering Committee. Inhibitor to factor VII in severe factor VII deficiency: detection and course of the inhibitory response. J Thromb Haemost 2005; 3 (04) 799-800
• 34 Giansily-Blaizot M, Aguilar-Martinez P, Biron-Andreani C, Jeanjean P, Igual H, Schved JF. Study Group of Factor Seven Deficiency. Analysis of the genotypes and phenotypes of 37 unrelated patients with inherited factor VII deficiency. Eur J Hum Genet 2001; 9 (02) 105-112
• 35 Landau D, Rosenberg N, Zivelin A, Staretz-Chacham O, Kapelushnik J. Familial factor VII deficiency with foetal and neonatal fatal cerebral haemorrhage associated with homozygosis to Gly180Arg mutation. Haemophilia 2009; 15 (03) 774-778
• 36 The European Association for Haemophilia and Allied Disorders (EAHAD). FVII gene (F7) gene variant database. Accessed February 17, 2021 at: http://www.factorvii.org/
• 37 Chollet ME, Andersen E, Skarpen E. et al. Factor VII deficiency: unveiling the cellular and molecular mechanisms underlying three model alterations of the enzyme catalytic domain. Biochim Biophys Acta Mol Basis Dis 2018; 1864 (03) 660-667
• 38 Harding HP, Calfon M, Urano F, Novoa I, Ron D. Transcriptional and translational control in the mammalian unfolded protein response. Annu Rev Cell Dev Biol 2002; 18: 575-599
• 39 Cooper DN, Millar DS, Wacey A, Banner DW, Tuddenham EG. Inherited factor VII deficiency: molecular genetics and pathophysiology. Thromb Haemost 1997; 78 (01) 151-160
• 40 Mariani G, Dolce A, Marchetti G, Bernardi F. Clinical picture and management of congenital factor VII deficiency. Haemophilia 2004; 10 (Suppl. 04) 180-183
• 41 Peyvandi F, Mannucci PM, Asti D, Abdoullahi M, DI Rocco N, Sharifian R. Clinical manifestations in 28 Italian and Iranian patients with severe factor VII deficiency. Haemophilia 1997; 3 (04) 242-246